The completion of the genome sequencing of humans and other species and the emergence of new technologies in mass spectrometry has together fostered unprecedented opportunities for studying proteins on a large scale. It is expected that large scale quantitative measurements of protein expression in different sets of samples, referred to as comparative proteomics, will advance our understanding of physiological processes and disease mechanisms. Comparative proteomic approaches have been applied to various biological samples to identify and quantify proteins that are up- or down-regulated in response to biological conditions. To date, there are two primary strategies used in current comparative proteomics; two dimensional gel electrophoresis (2D-PAGE) based strategy and mass spectrometry based in vitro stable isotope labeling strategy.
Although 2D-PAGE based methods have been a primary choice in comparative proteomics, 2D-gels are cumbersome to run, have a poor dynamic range, and are biased toward abundant and soluble proteins. In contrast, the mass spectrometry based stable isotope labeling strategy has a potential of overcoming most of the weaknesses of the 2D-PAGE based methods. If the stable isotope labeling can be achieved efficiently and equivalently for each distinct sample, then two samples may be compared using isotopic ratios. Among the in vitro stable isotope labeling methods, proteolytic 18O labeling is the simplest stable isotope labeling method and is expected to have the least methodological error (technical variations). Therefore, the proteolytic 18O labeling method has the potential to be a central method in comparative proteomics.
Currently, there are two ways to incorporate stable isotopes into peptides; first by derivatization of peptides by a light- or heavy-isotope coded reagent (Isotope Coded Affinity Tag or ICAT), or second by incorporation of 16O and 18O atom(s) into the carboxyl termini of peptides from the solvent water, H216O or H2 18O, respectively, upon proteolytic cleavage of proteins. The second method is referred to as a proteolytic 18O labeling method where a peptidase is used.
Although promising, a major drawback of the proteolytic 18O labeling method has been the generation of a mixture of isotopic isoforms upon proteolytic digestion resulting from the differential incorporation of either one or two 18O atoms (18O1/18O2) into each digested peptide species generated. Typically the serine proteases used include trypsin, Lys-C or Glu-C proteases. Unfortunately, past studies have found that the ratios of the first and the second 18O atom incorporation vary significantly with peptide sequences, and thus, the ratios of 18O1- and 18O2-peptides cannot be predicted with any certainty. The quantifications of the peptides results in significant errors in the calculations of 16O- and 18O-labeled peptide ratios. In spite of a more recent appreciation of this problem, no method has been reported to solve the problem.
Hydrolysis of a protein in H218O solvent by serine proteases may result in the incorporation of two 18O atoms into the carboxyl terminus of each proteolytically generated peptide provided that a sufficiently long time was given for the reaction to reach equilibrium. Studies done by Yao et al. demonstrated that the two 18O atoms are incorporated by trypsin at equilibrium via the following two step mechanism:RC16ONHR′+H218O→RC16O18O−++H3NR′  1)RC16O18O−+H218O→RC18O18O−+H216O  2)The first 18O atom is incorporated from the H218O solvent upon proteolytic cleavage of a peptide bond, as shown in reaction 1. The second 18O atom incorporation is essentially a carboxyl oxygen exchange reaction as shown in reaction 2, which occurs subsequent to proteolytic cleavage. The protease continues to interact with the peptide product and exchange the carboxyl oxygen, which results in two 18O atoms incorporation, if the first 18O atom is retained. However, reaction 2 is required to occur numerous times on the peptide to achieve complete incorporation of two 18O atoms. If the reaction continues, both oxygen atoms in the C-terminal carboxyl group of the peptide should theoretically come to equilibrium with oxygen from the H218O solvent. However, the reaction time required has proven to be not feasible and far less predictable than necessary to be utilized in labeling peptides for proteomics analysis.
In a recent invention (U.S. Patent Publication No. 2006/0105415) we reported that the incorporation of a single 18O atom can be accomplished using H218O solvent under conditions that optimize labeling based on reaction 1 upon proteolytic cleavage of a peptide bond while eliminating the slower rate limiting reaction 2. We demonstrated that at a higher pH reaction 1 could be optimized for incorporation of a single 18O atom upon protease cleavage and essentially no incorporation of a second 18O atom occurred. The conditions for various suitable proteases can be optimized to facilitate incorporation of a single 18O atom, avoid eliminating drawbacks previously employed for 18O labeling with peptidases, and provide highly accurate quantification method for comparative proteomics.
Complete double 18O atom incorporation has been more problematic to resolve because of the carboxyl oxygen exchange reaction. The carboxyl oxygen exchange reaction was found by Rittenberg and Sprinson for chymotrypsin fifty years ago. Shortly after their finding, Doherty and Vaslow demonstrated by an enzyme-substrate equilibrium experiment that the binding of acetyl-3,5-dibromo-L-tyrosine to chymotrypsin is stronger at acidic pH than alkaline pH. The kinetic parameters of chymotrypsin-catalyzed carboxyl oxygen exchange reaction have also been reported by Vaslow for acetyl-3,5-dibromo-L-tyrosine at pH 7.2 and by Bender and Kemp for benzoyl-L-phenylalanine and acetyl-L-tryptophane at pH 7.8. Recently, Yao and coworkers have reported kinetic parameters of trypsin-catalyzed carboxyl oxygen exchange reaction at pH 8.0 for short peptide substrates. However, low pH studies were not performed in these kinetic studies. A recent study done by Zang and coworkers showed that a trypsin-catalyzed carboxyl oxygen exchange reaction at pH 6.75 is more efficient than at pH 8.50 based on an experiment that measured the changes of isotopic peaks of the labeled peptides after 20 hours of labeling reactions. More recently, a study performed by Staes and coworkers in a lengthy three step process consisting of digestion with trypsin overnight, inactivation of trypsin by reductive alkylation, and finally overnight incubation at pH 4.5 with two 18O atoms reported that incorporation of two 18O atoms was achieved. However, these earlier works did not measure reaction rates or pH optima, therefore no quantitative information or optimal kinetic parameters on the rate of the reaction were obtained.